Methods for fabricating electrokinetic concentration devices

ABSTRACT

The present invention provides a device and methods of use thereof in concentrating a species of interest and/or controlling liquid flow in a device. The methods make use of a device comprising a fluidic chip comprising a planar array of channels through which a liquid comprising a species of interest can be made to pass with at least one rigid substrate connected thereto such that at least a portion of a surface of the substrate bounds the channels, and a high aspect ratio ion-selective membrane is embedded within the chip, attached to at least a portion of the channels. The device comprises a unit to induce an electric field in the channel and a unit to induce an electrokinetic or pressure driven flow in the channel.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 61/064,784, filedon 26 Mar. 2008 which is incorporated in their entirety herein byreference.

GOVERNMENT INTEREST STATEMENT

This invention was made in whole or in part with government supportunder EB005743 awarded by the National Institutes of Health and underCA119402 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This invention provides devices and methods of use thereof inconcentrating a charged species of interest in solution. This inventionprovides a concentration device, which is based on electrokinetictrapping of a charged species of interest, which can be further isolatedand analyzed.

BACKGROUND OF THE INVENTION

One of the major challenges of proteomics is the sheer complexity ofbiomolecule samples, such as blood serum or cell extract. Typical bloodsamples could contain more than 10,000 different protein species, withconcentrations varying over 9 orders of magnitude. Such diversity ofproteins, as well as their huge concentration ranges, poses a formidablechallenge for sample preparation in proteomics.

Conventional protein analysis techniques, based on multidimensionalseparation steps and mass spectrometry (MS), fall short because of thelimited separation peak capacity (up to ˜3000) and dynamic range ofdetection (˜10⁴). Microfluidic biomolecule analysis systems (so-calledμTAS) hold promise for automated biomolecule processing. Variousbiomolecule separation and purification steps, as well as chemicalreaction and amplification have been miniaturized on a microchip,demonstrating orders of magnitude faster sample separation andprocessing. In addition, microfluidic integration of two differentseparation steps into a multidimensional separation device has beendemonstrated. However, most microfluidic separation and sampleprocessing devices suffers from the critical issue of sample volumemismatch. Microfluidic devices are very efficient in handling andprocessing 1 pL˜1 nL of sample fluids, but most biomolecule samples areavailable or handled in a liquid volume larger than 1 μL. Therefore,microchip-based separation techniques often analyze only a smallfraction of available samples, which significantly limits the overalldetection sensitivity. In proteomics, this problem is exacerbated by thefact that information-rich signaling molecules (cytokines andbiomarkers, e.g.) are present only in trace concentrations (nM˜pMrange), and there is no signal amplification technique such aspolymerase chain reaction (PCR) for proteins and peptides.

What is needed is an efficient sample concentrator, which can taketypical sample volume of microliters or more and concentrate moleculesinto a smaller volume so that it can be separated and detected much moresensitively. Several strategies are currently available to providesample preconcentration in liquid, including field-amplified samplestacking (FAS), isotachophoresis(ITP), electrokinetic trapping, micellarelectrokinetic sweeping, chromatographic preconcentration, and membranepreconcentration. Many of these techniques are originally developed forcapillary electrophoresis, and require special buffer arrangementsand/or reagents. Efficiency of chromatographic and filtration-basedpreconcentration techniques depends on the hydrophobicity and the sizeof the target molecules.

Electrokinetic trapping is another means for such charged biomoleculeconcentration. When applying an electric field across an ion-selectivemembrane, a charge-depletion region is developed, which in combinationwith tangential flow (either pressure-driven or electroosmosis-driven),can concentrate the charged analytes inside a channel. Currently,however, the fabrication of such devices is cumbersome and complex,since the integration of sufficiently thin (˜5 um) ion-selectivemembranes into the device has been challenging. Thin Nafion membranesare easily breakable and handling requires extreme care since themembrane can be easily wrapped around itself, confounding planar devicefabrication methods.

Another attempt at planar devices sandwiched a thin ion-selectivemembrane between two planar microchips, each chip containing amicrochannel, however this led to imperfect sealing of the device,resulting in gap formation around the membrane and thereby currentleakage.

SUMMARY OF THE INVENTION

The invention provides, in one embodiment, a concentrating devicecomprising:

-   -   a fluidic chip comprising a planar array of channels through        which a liquid comprising a species of interest can be made to        pass;    -   at least one rigid substrate connected thereto such that at        least a portion of a surface of the substrate bounds the        channels;    -   a high aspect ratio ion-selective membrane, embedded within the        chip, attached to at least a portion of the channels;    -   a unit to induce an electric field in the channel; and    -   a unit to induce an electrokinetic or pressure driven flow in        the channel.

In one embodiment, the means for inducing an electric field in thechannel is a voltage supply, which in one embodiment is supplied atbetween 50 mV and 1500 V. In one embodiment, the voltage supply appliesequal voltage to opposing sides of said microchannels, or in anotherembodiment, the voltage supply applies greater voltage to one channel,as compared to another channel, or in another embodiment, the voltagesupply causes a potential difference between one area of saidmicrochannel, as compared to another area within said microchannel. Inanother embodiment the voltage supply creates a potential differencebetween at least two said channels.

In one embodiment, the width of the channel is between about 0.1-500 μm,and in one embodiment, the width of the channel is between about 10μm-200 μm. In some embodiments, the width of the channel is betweenabout 100-1200 μm. In some embodiments the depth of the channel isbetween about 0.5-200 μm, and in some embodiments, the depth of thechannel is between about 5-50 μm. In some embodiments the depth of thechannel is between about 50-150 μm. In some embodiments, theion-selective membrane has a width of between about 0.01-100 μm, and insome embodiments, the width of the ion-selective membrane is 1-10 μm. Insome embodiments, the ion-selective membrane has a width of betweenabout 100-500 nm. In some embodiments, the ion-selective membrane has adepth of between about 0.01-3000 μm, and in some embodiments, the depthof the ion-selective membrane is between about 10-500 μm and in someembodiments, the depth of the ion-selective membrane is between about100-1000 μm. In some embodiments, the ion-selective membrane has a depthof between about 550-1050 μm.

In some embodiments, the rigid substrate comprises pyrex, silicon,silicon dioxide, silicon nitride, quartz, PMMA, PC or acryl.

In one embodiment, the fluidic chip comprises polydimethylsiloxane.

In one embodiment, the high aspect ratio ion-selective membranecomprises polytetrafluorethylenes (PTFEs), perfluorosulfonates,polyphosphazenes, polybenzimidazoles (PBIs), poly-zirconia,polyethyleneimine-poly(acrylic acid), poly(ethylene oxide)-poly(acrylicacid), or non-fluorinated hydrocarbon polymers or polymer-inorganiccomposites. In some embodiments the high aspect ratio ion selectivemembrane comprises sulfonated tetrafluorethylene copolymer. In someembodiments the sulfonated tetrafluorethylene copolymer comprises Nafionor Nafion solution. In some embodiments the high aspect ratio ionselective membrane comprises microparticles or beads. In someembodiments the microparticles or beads comprises silica or polystyrene.

In some embodiments, the surface of the microchannel has beenfunctionalized to reduce or enhance adsorption of said species ofinterest to said surface, or in some embodiments, the surface of themicrochannel has been functionalized to enhance or reduce the operationefficiency of the device.

In some embodiments the high aspect ratio ion selective membrane is notin contact or is in minimal contact with the rigid substrate or thecover glass of the device.

In some embodiments, the unit to induce an electric field in the channelcomprises at least a pair of electrodes and a power supply. In someembodiments, the substrate comprises electrodes, which are positionedproximally to the ion-selective membrane.

In some embodiments, the device is coupled to a separation system,detection system, analysis system or combination thereof. In someembodiments, the device is coupled to a mass spectrometer.

In one embodiment, the invention provides for a method of concentratinga species of interest in a liquid, the method comprising applying aliquid comprising the species of interest to the devices of thisinvention. In one embodiment the device comprising

-   -   a fluidic chip comprising a planar array of channels through        which a liquid comprising a species of interest can be made to        pass;    -   at least one rigid substrate connected thereto such that at        least a portion of a surface of the substrate bounds the        channels;    -   a high aspect ratio ion-selective membrane, embedded within the        chip, attached to at least a portion of the channels;

In one embodiment, the method further comprises the steps of:

-   -   inducing an electric field in the channel whereby ion depletion        occurs in a region in the channel proximal to the high aspect        ratio ion-selective membrane embedded within the chip, and a        space charge layer is formed within the channel, which provides        an energy barrier to the species of interest; and    -   inducing liquid flow in the channel.

In one embodiment, the flow is electroosmotic, or in another embodiment,the flow is pressure driven.

In one embodiment, the steps are carried out cyclically.

In one embodiment, inducing an electric field in said channel is byapplying voltage to said device, which in one embodiment is between 50mV and 1500 V. In one embodiment, equal voltage is applied to opposingsides of the channel, or in another embodiment, greater voltage isapplied to the anodic side of the channel, as compared to the cathodicside.

In one embodiment, a space charge layer is generated in the channelprior to applying greater voltage to the anodic side of said channel.

In one embodiment, the liquid comprises an organ homogenate, cellextract or blood sample. In another embodiment, the species of interestcomprises proteins, polypeptides, nucleic acids, viral particles, orcombinations thereof.

According to this aspect of the invention and in another embodiment, thedevice is coupled to a separation system, detection system, analysissystem or combination thereof. In some embodiments the detection systemor analysis system comprises fluorescence. In one embodiment the highaspect ratio ion selective membrane is not in contact or is in minimalcontact with the rigid substrate. In some embodiments such minimal or nocontact eliminates or reduces contamination of the rigid substrate byfluorescent molecules. In some embodiments the concentration methodresults in large volumes of concentrated species in a liquid. In someembodiments the large concentrated volume containing the concentratedspecies is about 10 nL.

In some embodiments, this invention provides a method for thepreparation of a concentrating device comprising:

-   -   a fluidic chip comprising a planar array of channels through        which a liquid comprising a species of interest can be made to        pass;    -   at least one rigid substrate connected thereto such that at        least a portion of a surface of the substrate bounds the        channels;    -   a high aspect ratio ion-selective membrane, embedded within the        chip, attached to at least a portion of the channels;    -   the method comprising:    -   forming a high aspect ratio trench in said fluidic chip, such        that the trench is perpendicular to the long axis of the        channels, and such that the trench depth equals or exceeds the        depth of the channels in the fluidic chip;    -   bending the fluidic chip parallel to the long axis of the        trench, such that at least a portion of the trench becomes        wider;    -   applying a liquid polymer to an area proximal to one end of the        trench such that the liquid polymer is allowed to flow along the        trench and fill the trench.    -   unbending the fluidic chip such that the liquid polymer is        strongly adhered to the trench;    -   providing conditions such that the liquid polymer forms a high        aspect ratio ion selective membrane embedded in the trench; and    -   optionally removing residues of the polymer from areas of the        fluidic chip proximal to the trench;    -   attaching the rigid substrate to the fluidic chip comprising        channels such that the channels bound at least a portion of a        surface of the substrate.

In some embodiments, the liquid polymer comprisespolytetrafluorethylenes, polyphosphazenes, polybenzimidazoles (PBIs),poly-zirconia, polyethyleneimine-poly(acrylic acid), or poly(ethyleneoxide)-poly(acrylic acid). In some embodiments the liquid polymercomprises sulfonated tetrafluorethylene copolymer. In some embodimentsthe sulfonated tetrafluorethylene copolymer comprises a Nafion solutionor pure Nafion. In some embodiments the high aspect ratio ion selectivemembrane comprises microparticles or beads. In some embodiments themicroparticles or beads comprises silica or polystyrene.

In some embodiments the liquid polymer at least partially fills thechannels in an area proximal to the high aspect ratio ion selectivemembrane. In some embodiments the high aspect ratio ion selectivemembrane is not in contact or is in minimal contact with the rigidsubstrate. In some embodiments the invention provides for aconcentrating device made by the process of preparing a concentratingdevice of the present invention.

In one embodiment, high aspect ratio means that the depth is much largerthan the width. In one embodiment, high aspect ratio means that thedepth is of the order of hundreds to thousands of microns, while thewidth is of the order of single microns. In some embodiments, the highaspect ratio ion selective membrane has a width of between 0.1-100 μm.In some embodiments, the high aspect ratio ion selective membrane has awidth of between 1-6 μm. In some embodiments, the high aspect ratio ionselective membrane has a non uniform width. In some embodiments, thehigh aspect ratio ion selective membrane has a depth of between 10-1000μm. In some embodiments, the high aspect ratio ion selective membranehas a depth of between 500-850 μm. In some embodiments, the high aspectratio ion selective membrane has a depth of between 750-1250 μm. In oneembodiment, providing conditions such that the liquid polymer forms ahigh aspect ratio ion selective membrane embedded in the trench isaccomplished by heating the liquid polymer. In one embodiment heating isconducted at a temperature of about 95°c. In one embodiment heating isperformed for about 10 minutes. In one embodiment, attaching thesubstrate to the fluidic chip is by plasma bonding. In one embodiment,the length of the high aspect ratio membrane is any length that islonger than the gap between microchannels or reservoirs andmicrochannels of this invention.

In some embodiments, the fluidic chip comprising channels having a widthof between 10-200 μm. In some embodiments, the fluidic chip comprisingchannels having a depth of between 5-100 μm.

In some embodiments the rigid substrate comprises pyrex, silicon,silicon dioxide, silicon nitride, quartz, PMMA, PC or acryl. In someembodiments the fluidic chip comprises polydimethylsiloxane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts embodiments of methods for fabricating thedevices of the invention; PDMS microchannels are obtained from thestandard chip fabrication process (FIG. 1 a); a razor blade is used tocut across the microchannels for guiding Nafion infiltration afterpunching sample loading holes (FIG. 1 b); the depth of the cuttingreaches below the microchannel depth; by bending the chip, the gap wasopened and a drop of 1.5 μL Nafion 117 solution (Fluka) was loaded intothe loading holes (FIG. 1 c); The Nafion solution immediately fill boththe gap and a portion of the microchannels by capillary forces; After 10minutes of curing at 95° c, solvents in the Nafion resin evaporate andthe gap is bonded by the Nafion resin (FIG. 1 d); the elastic nature ofthe PDMS facilitates tight sealing of the Nafion in the gap; anyremaining Nafion on the top of the PDMS surface is removed by taping andpeeling, and vertical or planar type junctions are formed (inserts inFIG. 1 d); a glass plate is bonded on top of the device using plasmatreatment (FIG. 1 e).

FIG. 2 shows scanning electron microscope (SEM) images of a device ofthis invention. Three microchannels were connected to each other byself-sealing Nafion membrane structure (FIG. 2 a); FIG. 2 b is the SEMimage of a cross section (A-A′ in FIG. 2 a) of the membrane. Nafionresin was filled perpendicular to the plane of the cover glass and theNafion membrane thickness was estimated to be ˜2-4 μm (FIG. 2 b insert);FIG. 2 c is a current/voltage (I/V) plot for confirming repeatability.The ion current through the Nafion membrane was measured as a functionof voltage applied.

FIG. 3 depicts an embodiment of a preconcentration test in a device ofthe present invention. FIG. 3 a shows the preconcentration factors ofBODIPY disulfonate (Invitrogen) for three different concentrations (0.1nM, 1 nM and 10 nM) in 1 mM phosphate buffer solution using a doublegate (DG) device. The fluorescent intensities were measured and analyzedwith an inverted fluorescence microscope (IX-51) with a CCD camera(SensiCam, Cooke corp.) and Image Pro Plus 5.0 (Media Cybernetics inc.).Results show that preconcentration factors of up to 1×10⁴ are achievedwithin 15 minutes. The concentration factors depend on the operatingconditions such as applied voltage, charges of species, bufferconcentrations and microchannel dimensions. The preconcentration ofβ-phycoerythrin (β-PE) protein in the device is shown in FIG. 3 b fortwo initial concentrations of 1.67 nM and 16.7 pM. Concentration factorsof up to 1×10⁴ were achieved within 22 minutes.

FIG. 4 depicts an embodiment of the mechanism of concentration ofcharged species in a device of this invention using external pressurefields. An operation voltage of 120 V was applied at both reservoirs andthe external pressure flow was induced by a syringe pump (Harvard) at 35nL/min. The preconcentration speed (seven minutes) was doubled whencompared to the speed when no external pressure was applied (15minutes).

FIG. 5 depicts one embodiment of the pre-concentrator operating schemehigh performance nanofluidic pumping. Low concentrations of ions nearthe depletion zone result in a very high electric fields (up to ˜kV/cm).Thus, the flow inside the zone can push the sample liquid along themicrochannel. This flow can reverse the pressure flow as seen in FIG. 5a. The graph in FIG. 5 b. show the flow reverse process. Voltage wasincreased at a rate of 50 V/30 sec up to 300 V. The mechanism shown canbe used as a pump and can also be used for flow switching and gating.

FIG. 6 is a plot of depicts one embodiment of the pre-concentratoroperating scheme for a semi-macrochannel. A large macrochannel with thedimensions of 1000 Mm (width)×100 Mm (depth) was fabricated. Ioncurrents through the Nafion membrane were approximately 10 times higherthan ion currents in a channel with the dimensions of 100 Mm (width)×10Mm (depth) (compare FIG. 2 c to FIG. 6 a). Ion depletion in a singlegate (SG) device is shown in FIG. 6 b. Ion preconcentration in a DGdevice is shown in FIG. 6 c. The volume of the preconcentration plug wasnearly 10 nL which has never been demonstrated before. This device issuitable for connection to commercial analytical systems such as massspectrometry (MS) and matrix-assisted laser desorption/ionization(MALDI).

function of increasing trapping time. The data are shown for 10 mMphosphate buffer, pH=7 solution.

FIG. 7 depicts an embodiment of a device preparation procedure in whicha trench is patterned in the PDMS chip and filled with Nafion. TheNafion is cured and sealed within the PDMS gap. A glass substrate isplaced on top of the PDMS fluidic chip.

FIG. 8 is a device image of a microbead packed device fabricated usingthe self-sealed membrane method. The microbeads are polystyrene beadswith 1 mm diameter. The gap between the beads is approximately 40 nm.

FIG. 9 depicts the preconcentration of a FITC dye in a device comprisinga microbead packed high aspect ratio ion selective membrane.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides, in one embodiment, a concentrating device andmethods of use thereof, in concentrating a species of interest.

In some embodiments, this invention provides devices for concentrationand/or pre-concentration of a substance on a micro- or nano-scale. Thedevices of this invention, in some embodiments, make use of high aspectratio ion-selective membranes such as Nafion membranes, placed inmicrofluidic chips, through a unique fabrication process, which enables,in some embodiments, specific deposit of the ion-selective membrane in adevice, in a manner, which is inexpensive and promotes ready depositionand high stability despite the known fragility of such membranes tophysical manipulations, which in the past made their incorporation intosuch devices difficult.

In some embodiments, the devices and methods of this invention entaildepositing, flowing or infiltrating resin solutions and curing suchsolutions to form the ion selective membranes, as herein described. Insome embodiments, using the resin solution enables thin verticalmembrane formation on a fluidic chip, and incorporation of the sameadjacent to or in proximity to a microchannel of a device.

The invention provides, in one embodiment, a concentrating devicecomprising:

-   -   a fluidic chip comprising a planar array of channels through        which a liquid comprising a species of interest can be made to        pass;    -   at least one rigid substrate connected thereto such that at        least a portion of a surface of the substrate bounds the        channels;    -   a high aspect ratio ion-selective membrane, embedded within the        chip, attached to at least a portion of the channels;    -   a unit to induce an electric field in the channel; and    -   a unit to induce an electrokinetic or pressure driven flow in        the channel.

In some embodiments, this invention provides a method for thepreparation of a concentrating device comprising:

-   -   a fluidic chip comprising a planar array of channels through        which a liquid comprising a species of interest can be made to        pass;    -   at least one rigid substrate connected thereto such that at        least a portion of a surface of the substrate bounds the        channels;    -   a high aspect ratio ion-selective membrane, embedded within the        chip, attached to at least a portion of the channels;    -   the method comprising    -   forming a high aspect ratio trench in said fluidic chip, such        that the trench is perpendicular to the long axis of the        channels, and such that the trench depth equals or exceeds the        depth of the channels in the fluidic chip;    -   bending the fluidic chip parallel to the long axis of the        trench, such that at least a portion of the trench becomes        wider;    -   applying a liquid polymer to an area proximal to one end of the        trench such that the liquid polymer is allowed to flow along the        trench and fill the trench.    -   unbending the fluidic chip such that the liquid polymer is        strongly adhered to the trench;    -   providing conditions such that the liquid polymer forms a high        aspect ratio ion selective membrane embedded in the trench; and    -   optionally removing residues of the polymer from areas of the        fluidic chip proximal to the trench;    -   attaching the rigid substrate to the fluidic chip comprising        channels such that the channels bound at least a portion of a        surface of the substrate.

According to this aspect of the invention, and in one embodiment, thisinvention provides a device fabricated according to the precedingmethod.

In some embodiments, the liquid polymer comprisespolytetrafluorethylenes, polyphosphazenes, polybenzimidazoles (PBIs),poly-zirconia, polyethyleneimine-poly(acrylic acid), or poly(ethyleneoxide)-poly(acrylic acid).

In some embodiments, providing conditions such that the liquid polymerforms a membranous structure embedded within the chip is accomplished byheating the liquid polymer. In some embodiments the liquid polymer isheated to a temperature of about 95° c. In some embodiments the liquidpolymer is heated for 10 minutes.

In one embodiment, attaching the substrate to the fluidic chip is byplasma bonding.

In one embodiment, the invention provides various methods for patterningan ion-selective membrane on a rigid substrate, to form the devices ofthis invention. Such methods are described herein, and exemplified inexample 1 herein below.

In some embodiments, the patterning methods of this invention, anddevices made thereby comprise, inter alia, flowing a resin through amicro- or nano-channel in a device under negative pressure, flushing theresin, and curing the adhered thin layer which in turn forms a membranestructure. In some embodiments, the viscosity of the resin is varied, orin some embodiments, the pressure applied is varied, which in turn willaffect the thickness of the membrane formed thereby.

In some embodiments, the resin viscosity is varied, or in someembodiments, the hydrophobicity of the resin is varied, to affect thesubsequent width of the ion-selective membrane formed thereby.

In some embodiments, curing the liquid polymer comprises, irradiationwith UV light.

In some embodiments, the methods for producing micro- or nano-fluidicdevices with high-aspect-ratio, ion-selective membranes of thisinvention, may comprise, inter alia, use of two oppositely chargedpolyelectrolytes such as PSS/PAA, which acts as s supporting solidmatrix. Ion selectivity may then be imparted to the supporting matrix byinfiltrating the membrane with a resin, which imparts such properties,for example, infiltrating the membrane with Nafion resin. According tothis aspect of the invention and in one embodiment, due to the capillaryforce of the membrane, the pores of the polyelectrolyte membrane fillwith the Nafion resin imparting to the membrane ion selectivity. In someembodiments, removing excess Nafion resin residue from the channel isaccomplished by flushing the channel with deionized water.

It is to be understood that any liquid resin, or liquid polymer or asolution of monomers capable of polymerizing which when patterned andcured according to the methods as described herein, produces anion-selective membrane is to be considered as part of this invention,and the invention is not to be limited to the examples of constituentsof such resins as herein described.

In some embodiments, such membranes can be constructed so as to comprisea perfluorosulfonated membrane comprised of apolytetrafluoroethylene(PTFE)-crosslinked hydrophobic backboneimpregnated with hydrophilic sulfonic acid sites. In some embodiments,hydrocarbon polymer non-fluorinated, and polymer-inorganic compositemembranes can be similarly prepared, and used in the methods of thisinvention.

In some embodiments, the membranes/resins will comprise polymers such aspolyphosphazenes, polybenzimidazoles (PBIs), and/or zirconia-polymergels. In some embodiments the membrane comprises polyethyleneimine,polyacrylic acid, perfluorosulfonates, non-fluorinated hydrocarbonpolymers, polymer-inorganic composites, polyethylene oxide or poroussilica or alumina polymers.

In some embodiments, polyelectrolyte multilayer systems such as LPEI/PAAor PEO/PAA (LPEI: linear polyethyleneimine, PAA:poly(acrylic acid); PEO:poly(ethylene oxide)) may be used to form the membrane. In someembodiments, films constructed from LPEI and PAA exhibit an ionicconductivity as high as 10⁻⁵ S/cm⁻¹ at 100% relative humidity and roomtemperature, and thus are useful in the devices of this invention.

In some embodiments, a membrane of PEO and PAA can be constructed viahydrogen-bonding interactions, films with conductivities from 10⁻⁵ to ashigh as 10⁻⁴ S/cm⁻¹ at ambient conditions may be obtained. In someembodiments, the method comprising flowing a resin through a micro- ornano-channel in a device under negative pressure, flushing the resin,and curing the adhered thin layer is useful for producing the desiredion-selective membrane in the devices of this invention, usingpolyelectrolyte multilayers as described hereinabove.

In some embodiments, unique to the methods and devices of this inventionis the absence of a requirement for the physical manipulation of fragilemembranes in order to integrate such membranes into the devices of thisinvention. In some embodiments, the invention comprises processes forpatterning/depositing a resin into a trench in the fluidic chip followedby curing of the resin to form a membrane, which in turn is readilyintegrated in the device without further physical manipulation of theformed membrane. In some embodiments, the devices of this invention andprocesses for preparing the same comprise curing a resin to form themembrane, as part of the construction of the device, and makes use ofmaterials which are disposable, thus providing a simply manufactureddevice, which can readily be mass produced, to form arrays of parallelconcentrators on a medium that can be disposable.

In some embodiments, the methods for producing micro- or nano-fluidicdevices with ion-selective membranes of this invention, may comprise,the preparation of a high-aspect-ratio ion selective membrane, asexemplified in some embodiments herein. In some embodiments, such methodmay comprise building a high-aspect ratio membrane with amicrobead-based approach, as will be appreciated by one skilled in theart. Self-assembled colloidal particles may be infiltrated with a resin,for example, Nafion, as described herein. In some embodiments themicroparticles may be filtrated without a resin to form the membrane. Insome embodiments the microparticles are referred to as beads. In someembodiments the beads or microparticles comprises silica or polystyrene.In some embodiments the spaces between the packed beads form the poresneeded for fluid flow or for ion-passage. In some embodimentsmicroparticles or bead size is 1 micron. In some embodiments themicroparticle size is between 250 nm and 2 microns. In anotherembodiment, a trench, which is filled with the resin may be used tobuild the high-aspect ratio membrane. In one embodiment the trench isformed by cutting through the chip. In one embodiment the cut is doneusing a razor blade. In one embodiment the cut is done using a sharpobject. In one embodiment the cut is done using a needle. In oneembodiment the trench is made by lithography. In one embodiment thetrench is made by UV lithography and in another embodiment by e-beamlithography. In one embodiment the trench is done using etchingtechniques. In one embodiment etching is dry and in another embodimentetching is wet.

The invention provides concentrating or pre-concentrating devices. Insome embodiments, the concentrating device, which is referred to as a“concentrator”, in another embodiment, comprises at least onemicrochannel and/or at least one nanochannel, placed on a PDMS substratein a roughly planar format, wherein the channel is proximal to anion-selective membrane, and the channel is bounded by a rigid substrate.

In one embodiment, the fluidic chip comprising a planar array ofchannels through which a liquid comprising a species of interest can bemade to pass is formed using the technology of microfabrication andnanofabrication, for formation of the respective channels.

Microfabrication technology, or microtechnology or MEMS, in oneembodiment, applies the tools and processes of semiconductor fabricationto the formation of, for example, physical structures. Microfabricationtechnology allows one, in one embodiment, to precisely design features(e.g., wells, channels) with dimensions in the range of <1 mm to severalcentimeters on chips made, in other embodiments, of silicon, glass, orplastics. Such technology may be used to construct the microchannels ofthe concentrator, in one embodiment.

In another embodiment, construction of the microchannels of theconcentrator may be accomplished according to, or based upon any methodknown in the art, for example, as described in Z. N. Yu, P. Deshpande,W. Wu, J. Wang and S. Y. Chou, Appl. Phys. Lett. 77 (7), 927 (2000); S.Y. Chou, P. R. Krauss, and P. J. Renstrom, Appl. Phys. Lett. 67 (21),3114 (1995); Stephen Y. Chou, Peter R. Krauss and Preston J. Renstrom,Science 272, 85 (1996) and U.S. Pat. No. 5,772,905 hereby incorporatedherein, in their entirety, by reference. In one embodiment, themicrochannels can be formed by imprint lithography, interferencelithography, self-assembled copolymer pattern transfer, spin coating,electron beam lithography, focused ion beam milling, photolithography,reactive ion-etching, wet-etching, plasma-enhanced chemical vapordeposition, electron beam evaporation, sputter deposition, stamping,molding scanning probe techniques and combinations thereof. In someembodiments, the methods for preparation of the devices of thisinvention may comprise or be modifications of Astorga-Wells J. et al,Analytical Chemistry 75: 5207-5212 (2003); or Joensson, M. et al,Proceedings of the MicroTAS 2006 Symposium, Tokyo Japan, Vol. 1, pp.606-608. Alternatively, other conventional methods can be used to formthe microchannels.

In one embodiment, the microchannels are formed as described in J. Han,H. G. Craighead, J. Vac. Sci. Technol., A 17, 2142-2147 (1999) and J.Han, H. G. Craighead, Science 288, 1026-1029 (2000), hereby incorporatedfully herein by reference.

In one embodiment, a series of reactive ion etchings are conducted,after which nano- or micro-channels are patterned with standardlithography tools. In one embodiment, the etchings are conducted with aparticular geometry, which, in another embodiment, determines theinterface between the microchannels, and/or nanochannels. In oneembodiment, etchings, which create the microchannels, are performedparallel to the plane in which etchings for the nanochannels arecreated. In another embodiment, additional etching, such as, forexample, and in one embodiment, KOH etching is used, to produceadditional structures in the concentrator, such as, for example, forcreating loading holes.

In another embodiment, electrical insulation of the concentrator isaccomplished. In one embodiment, such insulation is accomplished vianitride stripping and thermal oxidation of the concentrator. In anotherembodiment, a surface of the concentrator, which in another embodimentis the bottom surface, may be affixed to a substrate, such as, forexample, and in one embodiment, a Pyrex wafer. In one embodiment, thewafer may be affixed using anodic bonding techniques.

In one embodiment, construction of the fluidic chip comprising a planararray of channels may be accomplished by methods known to one skilled inthe art, or adaptation of such methods, such as, for example thosedescribed in U.S. Pat. No. 6,753,200, fully incorporated herein byreference.

In one embodiment, the fabrication may use a shaped sacrificial layer,which is sandwiched between permanent floor and ceiling layers, with theshape of the sacrificial layer defining a working gap. When thesacrificial layer is removed, the working gap becomes a fluid channelhaving the desired configuration. This approach, in one embodiment,allows a precise definition of the height, width and shape of interiorworking spaces, or fluid channels, in the structure of a fluidic device.

The sacrificial layer is formed on a substrate, is shaped by a suitablelithographic process, for example, and is covered by a ceiling layer.Thereafter, the sacrificial layer may be removed with a wet chemicaletch, leaving behind empty spaces between the floor and ceiling layerswhich form working gaps which may be used as flow channels and chambersfor the concentrator. In such a device, the vertical dimension, orheight, of a working gap is determined by the thickness of thesacrificial layer film, which is made with precise chemical vapordeposition (CVD) techniques, and accordingly, this dimension can be verysmall.

In order to provide access to the sacrificial layer contained in thestructure for the etching solution, which is used to remove thesacrificial layer, one or more access holes may be cut through theceiling layer, with the wet etch removing the sacrificial layer throughthese holes. An extremely high etch selectivity may be required betweenthe sacrificial layer and the dielectric layers in order to allow theetch to proceed in the sacrificial layer a significant distancelaterally from the access holes without consuming the floor and ceilinglayers which compose the finished device. One combination of materials,which may be used for such a process is polysilicon and silicon nitride,for the sacrificial layer and for the floor and ceiling layers,respectively. Extremely high etch selectivities can be obtained withbasic solutions such as, in some embodiments, potassium hydroxide (KOH),sodium hydroxide (NaOH), or in another embodiment, tetramethyl ammoniumhydroxide (TMAH).

In some embodiments, the ceiling layer is the rigid substrate with whichthe ion-selective membrane is associated.

The access holes cut in the top layer may be covered, in anotherembodiment. For this purpose, a sealing layer of silicon dioxide may bedeposited on top of the ceiling lay to fill in the access holes, andthis additional thin film layer provides a good seal against leakage orevaporation of fluids in the working gap. SiO₂ CVD techniques, representother embodiments, which yield a low degree of film conformality, suchas very low temperature oxide (VLTO) deposition, form a reliable sealwithout excessive loss of device area due to clogging near the accessholes. If desired, the access holes may be drilled through the bottomlayer, instead of or in addition to the holes in the ceiling layer, andlater resealed by depositing a layer of silicon dioxide.

For example, in some embodiments, chemical vapor deposition (CVD) may beused to deposit the device materials, including permanent wallmaterials, which are usually a dielectric material such as siliconnitride or silicon dioxide, and nonpermanent sacrificial layermaterials, such as amorphous silicon or polysilicon.

In some embodiments, micro-channels and/or nano-channels are oriented inparallel on the chip, forming an array of channels, wherein each channelmay represent a concentrator, such that multiple parallel concentrationsmay be accomplished on a single chip. In some embodiments, the channelsintersect, such that material concentrated in a channel can, underappropriate conditions be conveyed to another concentrator on the chip,for example, post assay or exposure to a particular reagent. Accordingto this aspect, and in some embodiments, the array or channels, whichintersect, allow for multi-step concentration, for example followingmanipulation or exposure to a dilute environment, and repeatconcentration is desirable.

In some embodiments, the microchannels are positioned in any desiredorientation, for example as befitting to suit a particular purpose orcollection scheme, etc. axis of another.

In one embodiment, an interface region is constructed which connects thechannels on the chip, for example two microchannels of the concentratorof this invention. In one embodiment, diffraction gradient lithography(DGL) is used to form a gradient interface between the channels of thisinvention, where desired. In one embodiment, the gradient interfaceregion may regulate flow through the concentrator, or in anotherembodiment, regulate the space charge layer formed in the microchannel,which, in another embodiment, may be reflected in the strength ofelectric field, or in another embodiment, the voltage needed to generatethe space charge layer in the microchannel. In some embodiments, theion-selective membrane is positioned at such an interface.

In one embodiment, the gradient interface area is formed of lateralspatial gradient structures for narrowing the cross section of a valueon a desired scale, for example, from the micron to the nanometer lengthscale. In another embodiment, the gradient interface area is formed of avertical sloped gradient structure. In another embodiment, the gradientstructure can provide both a lateral and vertical gradient.

In one embodiment, the concentrating device may be fabricated bydiffraction gradient lithography, by forming a microchannel ormicrochannels on a substrate and forming a gradient interface areabetween the desired channels. The gradient interface area can be formed,in one embodiment, by using a blocking mask positioned above a photomask and/or photoresist during photolithography. The edge of theblocking mask provides diffraction to cast a gradient light intensity onthe photoresist.

In one embodiment, a concentrator may comprise a plurality of channels,including a plurality of microchannels, and/or a plurality ofnanochannels, or a combination thereof. In one embodiment, the phrase “aplurality of channels refers to more than two channels, or, in anotherembodiment, more than 5, or, in other embodiments, more than 10, 96,100, 384, 1,000, 1,536, 10,000, 100,000 or 1,000,000 channels, or in anynumber desired to suit a particular purpose. Similarly, arrangement ofthe channels on the chip may be so designed as to suit a particularapplication.

In one embodiment, a device may contain a plurality of high aspectratio, ion selective membranes. In one embodiment a plurality ofhigh-aspect ratio ion selective membranes can be fabricated on a chip.In one embodiment fabrication of a plurality of high-aspect ratio ionselective membranes may be done using multi-blade fabrication. In oneembodiment multi-blade fabrication can be used for commercialization ofa device containing a self-sealed membrane. In one embodiment aself-sealed membrane refers to the high aspect ratio ion selectivemembrane. In one embodiment “self-sealed” means that after infiltration,or passage or filling of the trench or the gap, or the scratch made bythe blade with a liquid polymer solution, unbending the chip andsolidifying the polymer causes a self-sealing process of the scratch orthe gap or the trench by the polymer.

In one embodiment, multi-blade fabrication can be used for massiveparallelization of the membrane or the device fabrication process. Inone embodiment, multi-blade fabrication can be used to make a pluralityof membranes in parallel. In one embodiment, multi-blade fabricationrenders the fabrication process fast. In one embodiment, multi-bladefabrication renders the device a low-cost device. In one embodimentmulti-blade fabrication is combined with multi-syringe or multidispenser system that enables parallel injection of liquid polymer toall trenches or cuts made by the multiple blades. In one embodiment themulti-blade fabrication technique is part of an automated fabricationtechnique, in which all steps of forming the high aspect ratio ionselective membranes are automated, and all steps are performed inparallel on many channels or on many device parts or on many devices. Inone embodiment such automation enables mass production of devices, lowcost, high yield and reproducibility of device properties. In oneembodiment parallel multi-blade fabrication facilitates quality controland reliability measurements to be done on selected devices. In oneembodiment multi-blade fabrication and/or automation of the process areachieved using computers, computer programs, robotics or a combinationthereof. In one embodiment the number of high aspect ratio ion selectivemembranes produced is equal to the number of channels described hereinabove. In one embodiment the number of high aspect ratio ion selectivemembranes produced is greater than the number of channels describedherein above. In one embodiment the number of high aspect ratio ionselective membranes produced is smaller than the number of channelsdescribed herein above. In one embodiment the number of high aspectratio ion selective membranes produced is more than 5, or, in otherembodiments, more than 10, 96, 100, 384, 1,000, 1,536, 10,000, 100,000or 1,000,000 channels, or in any number desired to suit a particularpurpose.

In one embodiment, the width of the microchannel is between 1-100 μm, orin another embodiment, between 1 and 15 μm, or in another embodiment,between 20 and 50 μm, or in another embodiment, between 25 and 75 μm, orin another embodiment, between 50 and 100 μm or in another embodiment,between 0.1 and 500 μm, or in another embodiment, between 10 and 25 μm,or in another embodiment, between 100 and 1200 μm. In one embodiment,the depth of the microchannel is between 0.5-50 μm, or in anotherembodiment, between 0.5 and 5 μm, or in another embodiment, between 5and 15 μm, or in another embodiment, between 10 and 25 μm, or in anotherembodiment, between 15 and 50 μm, or in another embodiment, between 1μm-50 μm, or in another embodiment, between 10 and 25 μm, or in anotherembodiment, between 15 and 40 μm, or in another embodiment, between 0.5and 200 μm. In another embodiment, the depth of the channel is between 1μm-50 μm, or in another embodiment, between 5 and 25 μm, or in anotherembodiment, between 5 and 50 μm, or in another embodiment, between 25and 50 μm, or in another embodiment between 50 and 150 μm.

In one embodiment, the concentrator is constructed as shown in FIG. 7 oraccording to the schematic provided in FIG. 1. The microchannels, areoriented in parallel on a PDMS chip (FIG. 1 a), and a cut is madeperpendicular to the long axis of the channels (FIG. 1 b). The PDMS chipis bent such that the gap formed by the cut is opened and become wider(FIG. 1 c). A drop of liquid polymer, microparticles, a solution thereofor a combination thereof is introduced proximal to one end of the gap(FIG. 1 d). A cover glass or another rigid substrate is plasma bonded tothe chip (FIG. 1 e). Photographs corresponding to the various processsteps are shown in FIG. 7.

In one embodiment the high aspect ratio ion selective membrane has awidth of between 0.01-100 μm. In one embodiment the high aspect ratioion selective membrane has a width of between 1-10 μm. In one embodimentthe high aspect ratio ion selective membrane has a width of between0.1-0.5 μm. In one embodiment the high aspect ratio ion selectivemembrane has a width of between 2-4 μm. In one embodiment the highaspect ratio ion selective membrane has a width of between 2-8 μm. Inone embodiment the high aspect ratio ion selective membrane has a widthof between 1-20 μm. In one embodiment the high aspect ratio ionselective membrane has a width of between 10-20 μm. In one embodimentthe high aspect ratio ion selective membrane has a width of between0.01-0.1 μm.

In one embodiment the high aspect ratio ion selective membrane has adepth of between 0.01-3000 μm. In one embodiment the high aspect ratioion selective membrane has a depth of between 10-500 μm. In oneembodiment the high aspect ratio ion selective membrane has a depth ofbetween 100-1000 μm. In one embodiment the high aspect ratio ionselective membrane has a depth of between 550-1050 μm. In one embodimentthe high aspect ratio ion selective membrane has a depth of between75-125 μm. In one embodiment the high aspect ratio ion selectivemembrane has a depth of between 1000-2000 μm. In one embodiment the highaspect ratio ion selective membrane has a depth of between 100-550 μm.In one embodiment the high aspect ratio ion selective membrane has adepth of between 200-800 μm or in another embodiment the ion selectivemembrane has a depth of 1-100 μm. In one embodiment the depth or widthor a combination thereof of the high aspect ratio ion selective membraneare non uniform. In one embodiment the depth of the membrane is measuredfrom the top of the PDMS chip and in one embodiment the depth of themembrane is measured from the bottom of the channels.

In some embodiments the chip comprises polydimethylsiloxane (PDMS).

In one embodiment the high aspect ratio ion selective membrane isembedded within the chip. In one embodiment “embedded” means confined,adhered to, placed between, shielded by, is part of, protected by,covered by or pressed between at least a portion of the chip. In someembodiments the membrane is formed by introducing a liquid polymer or asolution containing microparticles or nanoparticles to a trench in thechip. In some embodiments the polymer solution or polymer or a solutionor mixture of monomers or beads is cured within the trench. In someembodiments while curing the polymer or the membrane material, the twowalls of the trench are pressed together or are held tightly byreleasing from a bended orientation. In some embodiments curing themembrane material involves bond formation between the trench walls andthe membrane material. In some embodiments the bonds are covalent. Insome embodiments the bonds are polar or van der Waals bonds. In someembodiments the membrane material and the walls of the trench are heldtogether mechanically. In some embodiments the trench in the chip can bebent to open. In some embodiments bending widens the trench. In someembodiments bending results in a non-uniform width of the trench. Insome embodiments bending allows the easy passage of liquid membranematerial through the trench. In one embodiment the term “trench” refersto a slit, a gap, a space, a channel, or a hole, in the chip of thisinvention.

In one embodiment the high aspect ratio ion selective membrane isattached to at least a portion of the channels. In one embodiment themembrane is attached to the bottom and to the walls of the channels inthe area in which the cut or trench was made. In some embodiments whenthe membrane liquid material is infiltrated through the trench, aportion of it fills the channels in the area proximal to the trench. Insome embodiments residues of membrane material within the channels areremoved. In some embodiments the residues are removed by taping thesurface of the chip and peeling the tape. In some embodiments membranematerial on the surface of the chip and in the channels, adheres to thetape and is peeled off the chip when peeling the tape. In someembodiments the tape is a simple adhesive tape.

In one embodiment “high aspect ratio membrane” means a verticalmembrane. In one embodiment “high aspect ratio membrane” means amembrane in which the depth exceeds the width. In some embodiments thedepth is 25 folds larger than the width. In some embodiments the depthis 100 folds larger than the width. In some embodiments the depth is 250folds larger than the width. In some embodiments the depth exceeds thewidth by any amount that fits a certain application of the presentinvention. In some embodiments “ion selective” means that only a certaintype of ions can enter the membrane. In some embodiment ion selectiverefers to the size of the ions, the charge of the ions, shape or acombination thereof. In some embodiments ions can be organic orinorganic. In some embodiments the membrane is permeable to protons(positive hydrogen ions) only. In some embodiments ion-selective meansthat a membrane can pass ions and can prevent the passage or transfer ofuncharged species. In one embodiment the membrane is permeable topositive ions only, and is not permeable toward anions and electrons.

In one embodiment a method for the preparation of a device of thisinvention minimizes contact between the membrane material and the glassor cover substrate of the chip. In one embodiment only a thin portion ofthe membrane material touches the cover glass. In one embodiment suchconfiguration reduces the contact between contaminating molecules in thesample that are trapped in the membrane and the cover glass. In someembodiments, fluorescent molecules that are trapped or are passingthrough the membrane can interfere with a sample fluorescent signal, ifthe membrane area proximal to the cover glass is extensively large. Inone embodiment of the present invention, having the membrane embedded inthe chip as opposed to covering the glass, results in enhancedfluorescence signal resolution and clarity.

In another aspect of the invention, the concentrator further comprisesat least one sample reservoir in fluid communication with themicrochannel or microchannels. In another embodiment, the samplereservoir is capable of releasing a fluid or liquid comprising a speciesof interest. In one embodiment, the sample reservoir is connected to themicrochannel by means of a conduit, which may have the dimensions of themicrochannel, or may comprise a gradient interface area, as described.

In one embodiment, the introduction of a liquid comprising a species ofinterest in the device and independent induction of an electric field inthe nanochannel and/or in the microchannel, concentrates the species ofinterest within the channel.

In one embodiment, the concentrator makes use of an ion-selectivemembrane to generate ion-depletion regions for electrokinetic trapping,as exemplified and described herein.

In one embodiment, an electric field is applied to the concentrator andgenerates an ion-depletion region and extended space charge layer thattraps anionic molecules. A tangential field in the anodic side maygenerate electroosmotic flow, which draws molecules into a trappedregion.

In one embodiment, flow in the device may be pressure-driven, and may beaccomplished by any means well known to one skilled in the art. Inanother embodiment, the flow may be a hybrid of pressure-driven andelectrokinetic flow.

In one embodiment, the phrases “pressure-driven flow” refers to flowthat is driven by a pressure source external to the channel segmentthrough which such flow is driven, as contrasted to flow that isgenerated through the channel segment in question by the application ofan electric field through that channel segment, which is referred toherein, in one embodiment, as “electrokinetically driven flow.”

Examples of pressure sources include negative and positive pressuresources or pumps external to the channel segment in question, includingelectrokinetic pressure pumps, e.g., pumps that generate pressure byelectrokinetically driven flow in a pumping channel that is separatefrom the channel segment in question, provided such pumps are externalto the channel segment in question (see, U.S. Pat. Nos. 6,012,902 and6,171,067, each of which is incorporated herein by reference in itsentirety for all purposes).

In one embodiment, the term “electrokinetic flow” refers to the movementof fluid or fluid borne material under an applied electric field.Electrokinetic flow generally encompasses one or both ofelectrophoresis, e.g., the movement of charged species through themedium or fluid in which it is disposed, as well as electroosmosis,e.g., the electrically driven movement of the bulk fluid, including allof its components. Accordingly, when referred to in terms ofelectrokinetic flow, it will be appreciated that what is envisioned isthe full spectrum of electrokinetic flow from predominantly orsubstantially completely electrophoretic movement of species, topredominantly electroosmotically driven movement of material, e.g., inthe case of uncharged material, and all of the ranges and ratios of thetwo types of electrokinetic movement that fall between these extremes.

In one embodiment, reference to the term “liquid flow” may encompass anyor all of the characteristics of flow of fluid or other material througha passage, conduit, channel or across a surface. Such characteristicsinclude without limitation the flow rate, flow volume, the conformationand accompanying dispersion profile of the flowing fluid or othermaterial, as well as other more generalized characteristics of flow,e.g., laminar flow, creeping flow, turbulent flow, etc.

In one embodiment, hybrid flow may comprise pressure-based relay of theliquid sample into the channel network, followed by electrokineticmovement of materials, or in another embodiment, electrokinetic movementof the liquid followed by pressure-driven flow.

In one embodiment, the electric field may be induced in the respectivechannels by applying voltage from a voltage supply to the device. In oneembodiment voltage is applied by way of the placement of at least onepair of electrodes capable of applying an electric field across at leastsome of the channels in at least one direction. Electrode metal contactscan be integrated using standard integrated circuit fabricationtechnology to be in contact with at least one microchannel, or inanother embodiment, at least one nanochannel, or in another embodiment,a combination thereof, and oriented as such, to establish a directionalelectric field. Alternating current (AC), direct current (DC), or bothtypes of fields can be applied. The electrodes can be made of almost anymetal, and in one embodiment, comprise thin Al/Au metal layers depositedon defined line paths. In one embodiment, at least one end of oneelectrode is in contact with buffer solution in the reservoir.

In another embodiment, the concentrator may contain at least two pairsof electrodes, each providing an electric field in different directions.In one embodiment, field contacts can be used to independently modulatethe direction and amplitudes of the electric fields to, in oneembodiment, orient the space charge layer, or in another embodiment,move macromolecules at desired speed or direction, or in anotherembodiment, a combination thereof.

In one embodiment, the voltage applied is between 50 mV and 1500 V. Inone embodiment, the voltage supply applies equal voltage to opposingsides of the microchannel, or in another embodiment, the voltage supplyapplies greater voltage to the anodic side of said microchannel, ascompared to the cathodic side. In one embodiment, the voltage supplyapplies voltage across a microchannel. In one embodiment, the voltageacross the microchannel is applied such that the connection between themicrochannel and a nanochannel is in between the two points to whichvoltage is applied. In one embodiment, a higher positive voltage isapplied to one end of a microchannel and a lower positive voltage isapplied to the other end of the microchannel such that the connectionbetween the microchannel and a nanochannel is between these two ends. Inone embodiment, the end of the nanochannel that is farther away from themicrochannel is grounded. In one embodiment, 10 V are applied to one endof the microchannel, 5 V are applied to the other end of themicrochannel and the microchannel is connected to a nanochannel suchthat the end of the nanochannel that is far from the microchannel iselectrically grounded. In one embodiment, equal voltage is applied tothe two ends of the microchannel on two sides of the connection areabetween the nanochannel and the microchannel.

In one embodiment, instead of an array of microchannels, devices andmethods of this invention comprise only one microchannel and onenanochannel or only two microchannels and one nanochannel, or only twonanochannels and one microchannel, or any number of microchannels andnanochannels between one (1) and ten (10).

In one embodiment, the voltage supply may be any electrical source,which may be used to provide the desired voltage. The electrical sourcemay be any source of electricity capable of generating the desiredvoltage. For example, the electrical source may be a piezoelectricalsource, a battery, or a device powered by household current. In oneembodiment, a piezoelectrical discharge from a gas igniter may be used.

In one embodiment, the electrokinetic trapping in the device and samplecollection can occur over a course of minutes, or in another embodiment,can be maintained for several hours. In one embodiment, concentrationover a course of time results in concentration factors as high as10⁶-10⁸, and in another embodiment, may be even higher, uponoptimization of the conditions employed during the concentration, suchas by modifying the voltage applied, salt concentration of the liquid,pH of the liquid, ion-selective membrane choice of materials orthickness or combination thereof.

In another embodiment, the concentrator further comprises at least onewaste reservoir in fluid communication with the microchannel,microchannels, nanochannel and/or nanochannels of the concentrator. Inone embodiment, the waste reservoir is capable of receiving a fluid.

In one embodiment, the surface of the microchannel may be functionalizedto reduce or enhance adsorption of the species of interest to thesurface of the concentrator. In another embodiment, the surface of thenanochannel and/or microchannel has been functionalized to enhance orreduce the operation efficiency of the device. In another embodiment,external gate potential is applied to the substrate of the device, toenhance or reduce the operation efficiency of the device. In anotherembodiment, at least part of the device is comprised of a transparentmaterial. In another embodiment, the transparent material is pyrex,silicon dioxide, silicon nitride, quartz, PMMA, PC or acryl.

In another embodiment, the concentrator is adapted such that analysis ofa species of interest may be conducted, in one embodiment, in theconcentrator, or in another embodiment, downstream of the concentrator.In one embodiment, analysis downstream of the concentrator refers toremoval of the concentrated species from the device, and placement in anappropriate setting for analysis, or in another embodiment, constructionof a conduit from the concentrator which relays the concentratedmaterial to an appropriate setting for analysis. In one embodiment, suchanalysis may comprise signal acquisition, and in another embodiment, adata processor. In one embodiment, the signal can be a photon,electrical current/impedance measurement or change in measurements. Itis to be understood that the concentrating device of this invention maybe useful in various analytical systems, including bioanalysisMicrosystems, due to its simplicity, performance, robustness, andintegrability to other separation and detection systems, for example asdescribed herein below and depicted in FIG. 5. It is to be understoodthat any integration of the device into such a system is to beconsidered as part of this invention.

In another embodiment, the concentrator, or in another embodiment, themicrochannel or microchannels are capable of being imaged with atwo-dimensional detector. Imaging of the concentrator, or parts thereof,may be accomplished by presenting it to a suitable apparatus for thecollection of emitted signals, such as, in some embodiments, opticalelements for the collection of light from the microchannels.

In another embodiment, the device is coupled to a separation system, orin another embodiment, a detection system, or in another embodiment, ananalysis system or in another embodiment, a combination thereof. In oneembodiment the device is coupled to a mass spectrometer. In anotherembodiment, the device is coupled to an illumination source. Accordingto this aspect, and in some embodiments, assay of concentrated materialsmay be accomplished within devices as herein described, and theiranalysis may be affected by coupling appropriate detection apparatus andsystems to the device to conduct such analysis. In some embodiments,such assay may be enzymatic assay, probe detection of a desired product,synthetic procedures, digestion of materials, or others as will beappreciated by one skilled in the art. In some embodiment detection oranalysis is done using fluorescence techniques. In one embodiment afluorescent marker is bound to the species of interest. In oneembodiment an illumination source illuminates the channels containingthe species. In one embodiment fluorescence caused by illumination ofthe fluorescent marker is detected by a light detector. In someembodiments the concentration of the marker and of the species ofinterest can be measured quantitatively. In one embodiment suchmeasurement can detect the location and concentration of the species ofinterest. In some embodiments location and concentration of species ofinterest can be detected as a function of time.

In one embodiment, the concentrator may be disposable, and in anotherembodiment, may be individually packaged, and in another embodiment,have a sample loading capacity of 1-50,000 individual fluid samples. Inone embodiment, the concentrator can be encased in a suitable housing,such as plastic, to provide a convenient and commercially-readycartridge or cassette. In one embodiment, the concentrator will havesuitable features on or in the housing for inserting, guiding, andaligning the device, such that, for example, a sample loadingcompartment is aligned with a reservoir in another device, which is tobe coupled to the concentrator. For example, the concentrator may beequipped with insertion slots, tracks, or a combination thereof, orother adaptations for automation of the concentration process via adevice of this invention.

The concentrator may be so adapted, in one embodiment, for highthroughput screening of multiple samples, such as will be useful inproteomics applications, as will be appreciated by one skilled in theart.

In one embodiment, the concentrator is connected to electrodes, whichare connected to an electric potential generator, which may, in anotherembodiment be connected with metal contacts. Suitable metal contacts canbe external contact patches that can be connected to an externalscanning/imaging/electric-field tuner, in another embodiment.

In one embodiment of the present invention, the concentrator is a partof a larger system, which includes an apparatus to excite moleculesinside the channels and detect and collect the resulting signals. In oneembodiment, a laser beam may be focused upon the sample plug, using afocusing lens, in another embodiment. The generated light signal fromthe molecules inside the microchannels may be collected byfocusing/collection lens, and, in another embodiment, reflected off adichroic mirror/band pass filter into optical path, which may, inanother embodiment, be fed into a CCD (charge coupled device) camera.

In another embodiment, an exciting light source could be passed througha dichroic mirror/band pass filter box and focusing/collecting schemefrom the top of the concentrator. Various optical components and devicescan also be used in the system to detect optical signals, such asdigital cameras, PMTs (photomultiplier tubes), and APDs (Avalanchephotodiodes).

In another embodiment, the system may further include a data processor.In one embodiment, the data processor can be used to process the signalsfrom a CCD, to a digital image of the concentrated species onto adisplay. In one embodiment, the data processor can also analyze thedigital image to provide characterization information, such as sizestatistics, histograms, karyotypes, mapping, diagnostics information anddisplay the information in suitable form for data readout.

In one embodiment, the device is further modified to contain an activeagent in the microchannel. For example, and in one embodiment, themicrochannel is coated with an enzyme at a region wherein theconcentrated molecules will be trapped, according to the methods of thisinvention. According to this aspect, the enzyme, such as, a protease,may come into contact with concentrated proteins, and digest them.According to this aspect, the invention provides a method for proteomeanalysis, wherein, for example, a sample comprising a plurality ofcellular polypeptides is concentrated in the microchannel, to obtain aplurality of substantially purified polypeptides. The polypeptide isexposed to a protease immobilized within the microchannel, underconditions sufficient to substantially digest the polypeptide, therebyproducing digestion products or peptides. The digestion products may, inanother embodiment, then be transported to a downstream separationmodule where they are separated, and in another embodiment, from there,the separated digestion products may be conveyed to a peptide analysismodule. The amino acid sequences of the digestion products may bedetermined and assembled to generate a sequence of the polypeptide.Prior to delivery to a peptide analysis module, the peptide may beconveyed to an interfacing module, which in turn, may perform one ormore additional steps of separating, concentrating, and or focusing.

In other embodiments, the proteases include, but are not limited to:peptidases, such as aminopeptidases, carboxypeptidases, andendopeptidases (e.g., trypsin, chymotrypsin, thermolysin, endoproteinaseLys C, endoproteinase GluC, endoproteinase ArgC, endoproteinase AspN).Aminopeptidases and carboxypeptidases are useful in characterizingpost-translational modifications and processing events. Combinations ofproteases also can be used. In one embodiment, the proteases and/orother enzymes can be immobilized onto the microchannel surface usingadsorptive or covalent methods. In some embodiments, examples ofcovalent immobilization include direct covalent attachment of theprotease to a surface with ligands such as glutaraldehyde,isothiocyanate, and cyanogen bromide. In other embodiments, theproteases may be attached using binding partners which specificallyreact with the proteases or which bind to or react with molecules whichare themselves coupled to the proteases (e.g., covalently). Bindingpairs may include the following: cytostatin/papain,valphosphanate/carboxypeptidase A, biotin/streptavidin,riboflavin/riboflavin binding protein, antigen/antibody binding pairs,or combinations thereof.

In one embodiment, the steps of concentrating polypeptides obtained froma given cell, producing digestion products, and analyzing digestionproducts to determine protein sequence, can be performed in paralleland/or iteratively for a given sample, providing a proteome map of thecell from which the polypeptides were obtained. Proteome maps frommultiple different cells can be compared to identify differentiallyexpressed polypeptides in these cells, and in other embodiments, thecells may be subjected to various treatments, conditions, or extractedfrom various sources, with the proteome map thus generated reflectingdifferential protein expression as a result of the status of the cell.It is to be understood that such concentration and assay comprisemethods of this invention.

In some embodiments, the devices/methods of this invention may be usedto concentrate a desired material from a biological sample. In someembodiments, the biological sample may be a fluid. In one embodiment,such a fluid may comprise bodily fluids such as, in some embodiments,blood, urine, serum, lymph, saliva, anal and vaginal secretions,perspiration and semen, or in another embodiment, homogenates of solidtissues, as described, such as, for example, liver, spleen, bone marrow,lung, muscle, nervous system tissue, etc., and may be obtained fromvirtually any organism, including, for example mammals, rodents,bacteria, etc. In some embodiments, the solutions or buffered media maycomprise environmental samples such as, for example, materials obtainedfrom air, agricultural, water or soil sources, which are present in afluid which can be subjected to the methods of this invention. Inanother embodiment, such samples may be biological warfare agentsamples; research samples and may comprise, for example, glycoproteins,biotoxins, purified proteins, etc. In another embodiment, such fluidsmay be diluted.

In one embodiment, this invention provides an array architecture that iscapable of being scaled to at least 10,000 concentrators, suitable for areal-world screen.

In one embodiment, concentration efficiency may be determined by usinglabeled proteins or polypeptides, introduced into the concentrator inknown ratios and detecting the concentrated labeled protein orpolypeptides, such as exemplified herein below. Signal intensity can bedetermined as a function of time, over background noise.

In one embodiment, the concentrators of this invention may be undercontrolled physicochemical parameters, which may comprise temperature,pH, salt concentration, or a combination thereof.

In one embodiment, the invention provides for a method of concentratinga species of interest in a liquid, comprising using a device of theinvention, or one prepared by a process as herein described.

In one embodiment, the invention provides for a method of concentratinga species of interest in a liquid, the method comprising applying aliquid comprising the species of interest a device of this invention,the device comprising:

-   -   a fluidic chip comprising a planar array of channels through        which a liquid comprising a species of interest can be made to        pass;    -   at least one rigid substrate connected thereto such that at        least a portion of a surface of said substrate bounds said        channels; and    -   a high aspect ratio ion-selective membrane embedded within said        chip, attached to at least a portion of said channels.

In one embodiment, the method further comprises the steps of:

-   -   inducing an electric field in the channel whereby ion depletion        occurs in a region in the channel proximal to the high aspect        ratio ion-selective membrane, and a space charge layer is formed        within the channel, which provides an energy barrier to the        species of interest; and    -   inducing liquid flow in the channel.

In one embodiment, the flow is electroosmotic, or in another embodiment,the flow is pressure driven.

In one embodiment, the steps are carried out cyclically.

In one embodiment, inducing an electric field in said channel is byapplying voltage to said device, which in one embodiment is between 50mV and 1500 V. In one embodiment, equal voltage is applied to the twosides of the channel, or in another embodiment, greater voltage isapplied to the anodic side of the channel, as compared to the cathodicside.

In one embodiment, a space charge layer is generated in the channelprior to applying greater voltage to the anodic side of said channel.

According to this aspect of the invention and in another embodiment, thedevice is coupled to a separation system, detection system, analysissystem or combination thereof.

In one embodiment, the liquid is a solution. In another embodiment, theliquid is a suspension, which, in another embodiment is an organhomogenate, cell extract or blood sample. In one embodiment, the speciesof interest comprises proteins, polypeptides, nucleic acids, viralparticles, or combinations thereof. In one embodiment, the species ofinterest is a protein, nucleic acid, virus or viral particle found in,or secreted from a cell, and in another embodiment, is found in very lowquantities, such that it represents less than 10% of the proteinextracted form a protein extract of the cell.

In one embodiment, the methods of this invention and the devices of thisinvention enable collection of molecules from a relatively large (˜1 μLor larger) sample volume, and their concentration into a small (1 pL˜1nL) volume. Such concentrated sample can then, in other embodiments, beefficiently sorted, separated or detected by various microfluidicsystems, without sacrificing the overall detection sensitivity caused bythe small sample volume capacity of microfluidic biomoleculesorting/detection systems. In other embodiments the volume of theconcentrated species is large (˜10 nL).

In one embodiment, the methods and concentrating devices of thisinvention allow for significantly increased signal intensity of amolecules, and subsequent just detection, which, in another embodiment,allows for more aggressive molecular sorting and/or removal ofhigh-abundance molecules, such as proteins, from a sample, withoutsacrificing the detectability of molecules in minute concentration, suchas minor proteins or peptides.

In another embodiment, the devices for and methods of concentration ofthis invention enable the use of several non-labeling detectiontechniques (UV absorption, for example), which was not possible due tothe short path length and small internal volume of conventionalmicrofluidic channels. Therefore, in another embodiment, the devices forand methods of concentration of this invention, which combineconcentration and molecular sorting may provide an ideal platform forintegrated Microsystems for biomarker detection, environmental analysis,and chemical-biological agent detection.

In one embodiment, the method further comprises the step of releasingthe species of interest from the device. In one embodiment, the methodfurther comprises the step of subjecting the species of interest tocapillary electrophoresis.

Capillary electrophoresis is a technique that utilizes theelectrophoretic nature of molecules and/or the electroosmotic flow ofsamples in small capillary tubes to separate sample components.Typically a fused silica capillary of 100 μm inner diameter or less isfilled with a buffer solution containing an electrolyte. Each end of thecapillary is placed in a separate fluidic reservoir containing a bufferelectrolyte. A potential voltage is placed in one of the bufferreservoirs and a second potential voltage is placed in the other bufferreservoir. Positively and negatively charged species will migrate inopposite directions through the capillary under the influence of theelectric field established by the two potential voltages applied to thebuffer reservoirs. The electroosmotic flow and the electrophoreticmobility of each component of a fluid will determine the overallmigration for each fluidic component. The fluid flow profile resultingfrom electroosmotic flow is flat due to the reduction in frictional dragalong the walls of the separation channel. The observed mobility is thesum of the electroosmotic and electrophoretic mobilities, and theobserved velocity is the sum of the electroosmotic and electrophoreticvelocities.

In one embodiment of the invention, a capillary electrophoresis systemis micromachined onto a device, which is a part of, or separate from,the concentrating device described herein. Methods of micromachiningcapillary electrophoresis systems onto devices are well known in the artand are described, for example in U.S. Pat. No. 6,274,089; U.S. Pat. No.6,271,021; Effenhauser et al., 1993, Anal. Chem. 65: 2637-2642; Harrisonet al., 1993, Science 261: 895-897; Jacobson et al., 1994, Anal. Chem.66:1107-1113; and Jacobson et al., 1994, Anal. Chem. 66: 1114-1118.

In one embodiment, the capillary electrophoresis separations provide asample which may then be used for both MALDI-MS and/or ESI-MS/MS-basedprotein analyses (see, e.g., Feng et al., 2000, Journal of the AmericanSociety For Mass Spectrometry 11: 94-99; Koziel, New Orleans, La. 2000;Khandurina et al., 1999, Analytical Chemistry 71: 1815-1819.

In other embodiments, downstream separation devices, which may interfacewith the concentrator of this invention include, but are not limited to,micro high performance liquid chromatographic columns, for example,reverse-phase, ion-exchange, and affinity columns.

It is to be understood that the exact configuration of any systems,devices, etc. which are coupled downstream of the concentrating deviceare to be considered as part of this invention, and that theconfiguration may be varied, to suit a desired application. In oneembodiment, a module for separation of the concentrated peptides whichis positioned downstream of the concentrating device comprises aseparation medium and a capillary between the ends of which an electricfield is applied. The transport of a separation medium in the capillarysystem and the injection of the sample to be tested (e.g., a sample bandcomprising peptides and/or partially digested polypeptides) into theseparation medium can be carried out with the aid of pumps and valves,or in another embodiment, via electric fields applied to various pointsof the capillary.

In another embodiment, the method is utilized to detect said species ofinterest when said species is present in said liquid at a concentration,which is below a limit of detection.

In some embodiments concentration and assay of low abundance proteins isreadily accomplished with the devices/methods of this invention.Concentration of a low abundance protein of 10⁴ times was achieved in aslittle as 4 minutes, and a roughly 1000-fold enhancement in assaysensitivity was achieved, as compared to similar assay without using theconcentration methods/devices of this invention.

In other embodiments, various applications of the methods of the presentinvention are possible without deviating from the present invention.

By way of example, the concentrating and pumping methods of the presentinvention allow for high-throughput robotic assaying systems to directlyinterface with the devices of the present invention, and to concentratea species of interest, and/or and pump liquid.

Various modes of carrying out the invention are contemplated as beingwithin the scope of the following claims particularly pointing out anddistinctly claiming the subject matter, which is regarded as theinvention.

EXAMPLES Materials and Methods Device Fabrication:

Fabrication techniques for a microfluidic device comprising micro- ornano-channels were similar to those described (J. Han, H. G. Craighead,J. Vac. Sci. Technol., A 17, 2142-2147 (1999); J. Han, H. G. Craighead,Science 288, 1026-1029 (2000)). A PDMS device comprising microchannelswas fabricated.

A Nafion perfluorinated resin solution (5 wt. % in lower aliphaticalcohols and water containing 15-20% water) was used to form a highaspect ratio Nafion membrane embedded within the PDMS chip. The membranewas cured and integrated into the chip. The cover glass was adhered tothe chip by plasma bonding the PDMS chip containing the channels on topof the glass substrate.

An embodiment of a detailed scheme of the preparation of a device ofthis invention was carried out as follows: desired PDMS microchannelswere obtained from a standard PDMS chip fabrication process.Microchannels with various dimensions were fabricated. Dimensions ofchannels fabricated were 50 micron width×5 micron depth, 100 micronwidth×10 micron depth, 1000 micron width×100 micron depth. The centermicrochannel was connected to side channels through one side wall in asingle gate (SG) device and through both side walls in a double gate(DG) device. A mechanical cut was made across the microchannels using aconventional razor blade for guiding Nafion infiltration after punchingsample loading holes. The depth of the cutting was large enough to reachover the microchannel depth. The depth of the cut was typically 500-1000micron. Once the gap was created, PDMS tended to restore its inherentgeometric structure due to its flexibility. By bending the chip, the gapwas opened and a drop of 1.5 μL Nafion 117 solution (Fluka) was appliedto the edge of the gap. The Nafion solution immediately filled the boththe gap and a portion of the microchannels by capillary forces. Nafionis a sulfonated tetrafluorethylene copolymer, widely used as a protonconductor for proton exchange membranes. After 10 minutes of curing at95° c, solvents in the Nafion resin evaporated, and the gap was bondedby Nafion resin with the adhesive-assisting role of the Nafion resin.The elastic nature of PDMS seals the Nafion junction rather tightlybetween the PDMS walls in the gap. Any remaining Nafion resin on the topof the PDMS surface and in the channels was removed by taping andpeeling, and vertical type junctions were created within the PDMS,proximal to the microchannel bottom and proximal to the microchannelwalls. Finally, a glass plate was bonded on top of the device usingplasma treatment.

Biomolecule and Reagent Preparation

Molecules and dyes used included B-phycoerythrin, rGFP (BD bioscience,Palo Alto, Calif.), HTC-BSA (Sigma-Aldrich, St. Louis, Mo.),FITC-Ovalbumin (Molecular Probes, Eugene, Oreg.), HTC-BSA(Sigma-Aldrich, St. Louis, Mo.), HTC dye (Sigma-Aldrich, St. Louis,Mo.), Mito Orange (Molecular Probes, Eugene, Oreg.), and lambda-DNA (500μg/ml). DNA molecules were labeled with YOYO-1 intercalating dyes(Molecular Probles, Eugene, Oreg.) by following manufacturer'sinstruction

Scanning Electron Microscopy of Devices

The microscope image of fabricated junctions and of the microchannels ofDG devices were taken and are shown in FIG. 2. The images show threemicrochannels that were connected to each other by the high aspect ratioion selective membrane which was assembled across the microchannels.FIG. 2 b is an SEM image showing an image of a cross section (A-A′) ofthe device. Nafion resin was filled perpendicular to the plane of thecover glass and its thickness was estimated to be about 2-4 microns, asshown in the magnified image. Since the Nafion resin reached over 100microns in depth, it can be used as a “vertical type junction”. Voids inthe junction that could lead to unwanted leakage along the junctioncould not be seen. This vertical type Nafion junction had the dimensionsof ˜1000 micron depth×˜1 micron width. The cross sectional area of theformed junctions which is critical for high currents is theoretically atleast 1×10⁴ times larger than in the case of low aspect ratio or planarmembranes.

Repeatability Test

The DC ion current through the polymeric junction is an indicator fortesting reliability and repeatability of the performance of devices ofthe present invention. The initial ion currents were measured andcompared for different devices. The measurement was done using Keithley236 Current/Voltage Source-Measure Unit (Keithley Instruments, Inc.)which was connected to the SG device with microchannels dimensions of 50micron width×5 micron depth. In order to control the level of electricaldouble layer overlapping, 1 mM phosphate buffer was used. Four deviceswere fabricated and tested. Each device was tested three times. Thecurrent was proportional to the applied voltage and showed an excellentlinearity when the applied voltage was under 50 V. For most applicationsthe voltage does not exceed this limit. The results demonstrate thereliability and reproducibility of the fabrication method and of thedevices. The devices measured were compared to a device lacking theNafion membrane. In this device the current was less than 10 nA whichwas not sufficient to induce ion concentration polarization (iondepletion).

Optical Detection Setup

All the experiments were conducted on an inverted microscope (IX-51)with fluorescence excitation light source attached. A thermoelectricallycooled CCD camera (Cooke Co., Auburn Hill, Mich.) was used forfluorescence imaging. Sequences of images were analyzed by IPLab 3.6(Scanalytics, Fairfax, Va.). A home-made voltage divider was used todistribute different potentials to reservoirs. The built in 100 Wmercury lamp was used as a light source.

Channels were filled with 40 nM, 4 nM and 4 μM B-phycoerythrinsolutions, and the fluorescence intensity was determined. The camerashutter was opened only during periodical exposures (˜1 sec) to minimizephotobleaching of the collected molecules.

Example 1 Ion/Protein Concentration

The preconcentration factors of BODIPY disulfonate (Invitrogen) weremeasured using a device and methods of this invention. Three differentsolution concentrations were used. The concentrations used were 0.1 nM,1 nM and 10 nM, in a 1 mM phosphate buffer solution. A DG device wasused. Microchannels had dimensions of 100 microns width×10 micron depthas shown in FIG. 3 a. The average tangential electric field was 50 V/cm.The fluorescent intensities were measured and analyzed as described inthe method section herein above. Compared with its standard signalintensities (0.1 μM, 1 μM and 10 μM) the results showed thatpreconcentration factors of up to 1×10⁴ were achieved within 15 minutes.The preconcentration of β-phycoerythrin (β-PE) protein in the samedevice was measured. The preconcentration for two initial concentrations(1.67 nM and 16.7 pM) was measured and is shown in FIG. 3 b. The 1×10⁴preconcentration factors were achieved after 22 minutes. It wasconcluded that the concentration factors largely depend on the operatingconditions such as applied voltage, charges of target species, bufferconcentrations and the dimensions of the microchannel.

Example 2 Pressure Driven Ion/Protein Preconcentration Operation

Pressure driven preconcentration operations were conducted in devices ofthe present invention. FIG. 4 shows the preconcentration operation usingexternal pressure fields. Operation voltages of 120 V were applied atboth reservoirs causing a depletion voltage condition. External pressureflow was induced by a syringe pump (Harvard) at 35 nL/min from right toleft. This tangential pressure flow (35 nL/min corresponding to 2.6mm/sec in linear fluid velocity in this microchannel dimension) was atleast 20 times faster than the electrokinetic velocity that can beobtained by the tangential field of 50 V/cm used in FIG. 3. The speed ofpreconcentration was enhanced approximately 2× compared to the speedshown in FIG. 3. The preconcentration reached 1×10⁴ within 7 minutes inFIG. 4.

Example 3 High Performance Nanofluidic Pumping

The ionic concentration inside the ion depletion zone created near themembrane junction is so low that the solution can be considered desalted(below a few μM). In such a case the electric field inside this zoneincreases up to ˜kV/cm, even when an external electric field of 10 V/cmwas applied. Thus the flow inside the zone experienced extremely highelectric field and pushed the sample liquid all along the microchannel.This flow has reversed the pressure flow from opposite reservoir asshown in FIG. 5 a. A syringe pump was connected to the right centerreservoir with 0.1 μL/min flow. Initially, all liquid flew from right toleft without external electric field. While keeping the 0 V at the rightcenter reservoir, increasing the voltage at the left center reservoirinitiated a concentration polarization near the membrane. After acertain voltage (60 V) was reached, the ion depletion zone wassuccessfully created at the left hand side of the junction and it actedas a high performance pump. This operation can push the flow against theexternal pressure field as shown in FIG. 5 b.

Example 4 Ion Depletion and Preconcentration in a Semi Macro Channel

In order to demonstrate the efficiency of the high aspect ratio ionselective membrane devices, a larger microchannel was fabricated. Thechannel dimensions were 1000 μm width×100 μm depth. Since the deepermicrochannel gives higher ion current, in the high aspect ratioconfiguration ion current through the membrane was approximately 10times larger than in the smaller (100 μm width×10 μm depth) channel.This is shown in FIG. 6 a and is compared with FIG. 2 c. Due to thishigh ion current through the ion selective membrane, ion depletion wasdemonstrated in an SG device as shown in FIG. 6 b, and ionpreconcentration in a DG device as shown in FIG. 6 c. The volume of thepreconcentration plug was nearly 10 nL which was never demonstratedbefore and is suitable for applications in which the device is connectedto commercial analytical systems such as mass spectrometers and matrixassisted laser desorption/ionization (MALDI) instruments.

It will be understood by those skilled in the art that various changesin form and details may be made therein without departing from thespirit and scope of the invention as set forth in the appended claims.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed in the scope of the claims.

In the claims articles such as “a,”, “an” and “the” mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” or “and/or” betweenmembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention provides, in various embodiments, all variations,combinations, and permutations in which one or more limitations,elements, clauses, descriptive terms, etc., from one or more of thelisted claims is introduced into another claim dependent on the samebase claim unless otherwise indicated or unless it would be evident toone of ordinary skill in the art that a contradiction or inconsistencywould arise. Where elements are presented as lists, e.g., in Markushgroup format or the like, it is to be understood that each subgroup ofthe elements is also disclosed, and any element(s) can be removed fromthe group.

It should it be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in haec verba herein.

Certain claims are presented in dependent form for the sake ofconvenience, but Applicant reserves the right to rewrite any dependentclaim in independent format to include the elements or limitations ofthe independent claim and any other claim(s) on which such claimdepends, and such rewritten claim is to be considered equivalent in allrespects to the dependent claim in whatever form it is in (eitheramended or unamended) prior to being rewritten in independent format.

1. A concentrating device comprising: a fluidic chip comprising a planararray of channels through which a liquid comprising a species ofinterest can be made to pass; at least one rigid substrate connectedthereto such that at least a portion of a surface of said substratebounds said channels; a high aspect ratio ion-selective membrane,embedded within said chip, attached to at least a portion of saidchannels; a unit to induce an electric field in said channel; and a unitto induce an electrokinetic or pressure driven flow in said channel. 2.The device of claim 1, wherein said means for inducing an electric fieldin said channel is a voltage supply.
 3. The device of claim 2, whereinsaid voltage applied by said voltage supply is between 50 mV and 1500 V.4. The device of claim 2, wherein said voltage supply applies equalvoltage to opposing sides of said microchannel.
 5. The device of claim2, wherein said voltage supply applies greater voltage to the anodicside of said channel, as compared to the cathodic side.
 6. The device ofclaim 1, wherein the width of said channel is between 0.1-500 μm.
 7. Thedevice of claim 1, wherein the depth of said channel is between 5-50 μm.8. The device of claim 1, wherein the depth of said channel is between50-150 μm.
 9. The device of claim 1, wherein said rigid substratecomprises pyrex, silicon, silicon dioxide, silicon nitride, quartz,PMMA, PC or acryl.
 10. The device of claim 1, wherein said fluidic chipcomprises polydimethylsiloxane.
 11. The device of claim 1, wherein saidhigh aspect ratio ion-selective membrane comprisespolytetrafluoroethylenes (PTFEs), polyphosphazenes, polybenzimidazoles(PBIs), poly-zirconia, polyethyleneimine-poly(acrylic acid),perfluorosulfonates, non-fluorinated hydrocarbon polymers,polymer-inorganic composites or poly(ethylene oxide).
 12. The device ofclaim 1, wherein said liquid polymer comprises sulfonatedtetrafluorethylene copolymer.
 13. The device of claim 12, wherein saidsulfonated tetrafluorethylene comprises a Nafion solution.
 14. Thedevice of claim 1, wherein said high aspect ratio membrane comprisesmicroparticles or beads.
 15. The device of claim 14, wherein saidmicroparticles or beads comprising silica or polystyrene.
 16. The deviceof claim 1, wherein said high aspect ratio ion-selective membrane has awidth of 1-10 μm.
 17. The device of claim 1, wherein said ion-selectivemembrane has a depth of 550-1050 μm.
 18. The device of claim 1, whereina surface of said microchannel has been functionalized to reduce orenhance adsorption of said species of interest to said surface.
 19. Thedevice of claim 1, wherein the surface of the microchannel has beenfunctionalized to enhance or reduce the operation efficiency of thedevice.
 20. The device of claim 1, wherein said high aspect ratio ionselective membrane is not in contact or is in minimal contact with saidrigid substrate.
 21. The device of claim 1, wherein said unit to inducean electric field in said channel comprises at least a pair ofelectrodes and a power supply.
 22. The device of claim 1, wherein saiddevice is coupled to a separation system, detection system, analysissystem or combination thereof.
 23. The device of claim 1, wherein thedevice is coupled to a mass spectrometer.
 24. A method of concentratinga species of interest in a liquid, the method comprising applying saidliquid comprising said species of interest to a concentrating devicecomprising: a fluidic chip comprising a planar array of channels throughwhich a liquid comprising a species of interest can be made to pass; atleast one rigid substrate connected thereto such that at least a portionof a surface of said substrate bounds said channels; and a high aspectratio ion-selective membrane embedded within said chip, attached to atleast a portion of said channels.
 25. The method of claim 24, furthercomprising the steps of: inducing an electric field in said channelwhereby ion depletion occurs in a region in said channel proximal tosaid high aspect ratio ion-selective membrane, and a space charge layeris formed within said channel, which provides an energy barrier to saidspecies of interest; and inducing liquid flow in said channel.
 26. Themethod of claim 25, wherein said flow is electroosmotic.
 27. The methodof claim 25, wherein said flow is pressure driven.
 28. The method ofclaim 25, wherein steps are carried out cyclically.
 29. The method ofclaim 25, wherein inducing an electric field in said channel is byapplying voltage to said device.
 30. The method of claim 29, whereinsaid voltage is between 50 mV and 1500 V.
 31. The method of claim 29,wherein equal voltage is applied to opposing sides of said channel. 32.The method of claim 29, wherein greater voltage is applied to the anodicside of said channel, as compared to the cathodic side.
 33. The methodof claim 32, wherein a space charge layer is generated in said channelprior to applying said greater voltage to said anodic side of saidchannel.
 34. The method of claim 24, wherein said liquid comprises anorgan homogenate, cell extract or blood sample.
 35. The method of claim24, wherein said species of interest comprises proteins, polypeptides,nucleic acids, viral particles, or combinations thereof.
 36. The methodof claim 24, wherein said device is coupled to a separation system,detection system, analysis system or combination thereof.
 37. The methodof claim 36, wherein said detection system comprises fluorescence. 38.The method of claim 24, wherein said high aspect ratio ion selectivemembrane is not in contact or is in minimal contact with said rigidsubstrate.
 39. The method of claim 38, wherein said no contact orminimal contact eliminates or reduces contamination of said rigidsubstrate by fluorescent molecules.
 40. The method of claim 24, whereinsaid concentrating results in large concentrated volumes of said speciesof interest.
 41. The method of claim 40, wherein said large concentratedvolume of said species of interest is about 10 nL.
 42. A method for thepreparation of a concentrating device comprising: a fluidic chipcomprising a planar array of channels through which a liquid comprisinga species of interest can be made to pass; at least one rigid substrateconnected thereto such that at least a portion of a surface of saidsubstrate bounds said channels; and a high aspect ratio ion-selectivemembrane embedded within said chip, attached to at least a portion ofsaid channels; said method comprising: forming a high aspect ratiotrench in said fluidic chip, such that the trench is perpendicular tothe long axis of said channels, and such that the trench depth equals orexceeds the depth of said channels in said fluidic chip; bending saidfluidic chip parallel to the long axis of said trench, such that atleast a portion of said trench becomes wider; applying a liquid polymerto an area proximal to one end of said trench such that the liquidpolymer is allowed to flow along the trench and fill the trench.unbending said fluidic chip such that said liquid polymer is stronglyadhered to said trench; providing conditions such that said liquidpolymer forms a high aspect ratio ion selective membrane embedded insaid trench; and optionally removing residues of said polymer from areasof said fluidic chip proximal to the trench; attaching said one rigidsubstrate to said fluidic chip comprising channels such that saidchannels bound at least a portion of a surface of said substrate. 43.The method of claim 42, wherein said fluidic chip comprises channelshaving a width of between 10-200 μm.
 44. The method of claim 42, whereinsaid fluidic chip comprises channels having a depth of between 5-100 μm.45. The method of claim 42, wherein said high aspect ratio ion selectivemembrane has a width of between about 0.1-100 μm.
 46. The method ofclaim 42, wherein said high aspect ratio ion selective membrane has awidth of between about 1-6 μm.
 47. The method of claim 42, wherein saidhigh aspect ratio ion selective membrane has a non-uniform width. 48.The method of claim 42, wherein said high aspect ratio ion selectivemembrane has a depth of between about 10-1000 μm.
 49. The method ofclaim 42, wherein said high aspect ratio ion selective membrane has adepth of between about 500-850 μm.
 50. The method of claim 42, whereinsaid high aspect ratio ion selective membrane has a depth of betweenabout 750-1250 μm.
 51. The method of claim 42, wherein said rigidsubstrate comprises pyrex, silicon, silicon dioxide, silicon nitride,quartz, PMMA, PC or acryl.
 52. The method of claim 42, wherein saidfluidic chip comprises polydimethylsiloxane.
 53. The method of claim 42wherein said liquid polymer comprises polytetrafluoroethylenes,polyphosphazenes, polybenzimidazoles (PBIs), poly-zirconia,polyethyleneimine-poly(acrylic acid), or poly(ethyleneoxide)-poly(acrylic acid).
 54. The method of claim 42, wherein saidliquid polymer comprises sulfonated tetrafluorethylene copolymer. 55.The method of claim 54, wherein said sulfonated tetrafluorethylenecopolymer comprises a Nafion solution.
 56. The method of claim 42,wherein said high aspect ratio membrane comprises microparticles orbeads.
 57. The method of claim 56, wherein said microparticles or beadscomprising silica or polystyrene.
 58. The method of claim 42, whereinproviding conditions such that said liquid polymer forms a high aspectratio ion selective membrane embedded in said trench is accomplished byheating said liquid polymer.
 59. The method of claim 58, wherein saidheating is conducted at a temperature of about 95° c.
 60. The method ofclaim 58, wherein said heating is performed for about 10 minutes. 61.The method of claim 42, wherein attaching said substrate to said fluidicchip is done by plasma bonding.
 62. The method of claim 42, wherein saidliquid polymer at least partially fills said channels in an areaproximal to said high aspect ion selective membrane.
 63. The method ofclaim 42, wherein said high aspect ratio ion selective membrane is notin contact or is in minimal contact with said rigid substrate.
 64. Aconcentrating device made by the process of claim 42.